Anti-TFPI antibody (ab66544)

Submit a review Q&A (3)References (2)

Overview

  • Product name

    Anti-TFPI antibody
    See all TFPI primary antibodies

  • Description

    Rabbit polyclonal to TFPI

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, IHC-Frmore details

  • Species reactivity

    Reacts with: Human
    Predicted to work with: Rat, Cow, Chimpanzee, Rhesus monkey, Orangutan

  • Immunogen

    Synthetic peptide corresponding to Human TFPI aa 1-100 conjugated to keyhole limpet haemocyanin.
    (Peptide available as ab66543)

  • Positive control

    • This antibody gave a positive signal in the following Human Whole Cell Lysates: HeLa, Jurkat, A431, HEK293, HepG2, MCF7, SHSY-5Y and U2OS.

Properties

  • Form

    Liquid

  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.

  • Storage buffer

    pH: 7.40
    Preservative: 0.02% Sodium azide
    Constituent: PBS

    Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help.
  • Concentration information loading...

  • Purity

    Immunogen affinity purified

  • Clonality

    Polyclonal

  • Isotype

    IgG

  • Research areas

Applications

Our Abpromise guarantee covers the use of ab66544 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB Use a concentration of 1 µg/ml. Detects a band of approximately 52 kDa (predicted molecular weight: 35 kDa).
IHC-Fr Use at an assay dependent concentration. PubMed: 24716194

Target

  • Function

    Inhibits factor X (X(a)) directly and, in a Xa-dependent way, inhibits VIIa/tissue factor activity, presumably by forming a quaternary Xa/LACI/VIIa/TF complex. It possesses an antithrombotic action and also the ability to associate with lipoproteins in plasma.

  • Tissue specificity

    Mostly in endothelial cells.

  • Sequence similarities

    Contains 3 BPTI/Kunitz inhibitor domains.

  • Domain

    This inhibitor contains three inhibitory domains. The first domain interacts with VIIa and TF, the second one with Xa.

  • Post-translational
    modifications

    O-glycosylated.

  • Cellular localization

    Secreted.
  • Information by UniProt

  • Database links

    see all

  • Alternative names

    • Anti convertin antibody
    • EPI antibody
    • Extrinsic pathway inhibitor antibody
    • LACI antibody
    • Lipoprotein associated coagulation inhibitor antibody
    • Lipoprotein-associated coagulation inhibitor antibody
    • TFI antibody
    • TFPI 1 antibody
    • TFPI antibody
    • TFPI1 antibody
    • TFPI1_HUMAN antibody
    • Tissue factor pathway inhibitor (lipoprotein associated coagulation inhibitor) antibody
    • Tissue factor pathway inhibitor antibody
    see all

Images

  • Western blot - Anti-TFPI antibody (ab66544)
    Western blot - Anti-TFPI antibody (ab66544)
    All lanes : Anti-TFPI antibody (ab66544) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
    Lane 3 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 4 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
    Lane 5 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
    Lane 6 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
    Lane 7 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate
    Lane 8 : U2OS (Human osteosarcoma cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Performed under reducing conditions.

    Predicted band size: 35 kDa
    Observed band size: 52 kDa
    why is the actual band size different from the predicted?



    This protein contains a number of potential glycosylation sites (SwissProt). The 52 kDa band observed is comparable to molecular weights seen with other commercially available antibodies to TFPI.

References

This product has been referenced in:

  • Cheng X  et al. Trimethylamine N-oxide promotes tissue factor expression and activity in vascular endothelial cells: A new link between trimethylamine N-oxide and atherosclerotic thrombosis. Thromb Res 177:110-116 (2019). Read more (PubMed: 30875490) »
  • Liczko J  et al. Tissue factor and tissue factor pathway inhibitor in chronically inflamed gallbladder mucosa. Biomed Res Int 2014:403639 (2014). IHC-Fr ; Human . Read more (PubMed: 24716194) »
See all 2 Publications for this product

Publishing research using ab66544? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-3 of 3 Abreviews or Q&A

Question

Thank you again for your reply and suggestions. I&apos;m currently
testing de-glycosylation of the samples and hopefully this will
show which of the bands or if both of them are correct.
The rTFPI control is from another company and it is
purified from cell cultures. It does not say from which cells but
that the weight should be about 35 kDa. Thus, I would think it is
not much glycosylated no.

Read More
Answer

Thank you for your reply.
Thank you for trying some of the tips. Please do let me know what the results are once you have them. I'm curious myself, as well as want to ensure the antibodies are working as expected.

Should it turn out that one or bothantibodies are not working as expected, and they were purchased within the last 6 months, we can discuss then other options of resolving the issue.

I wish you good luck and look forward to hear back from you.

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Question

1) My samples are supernatants from Sum102 breast cancer cells
treated with PI-PLC (2 hours) to cleave off GPI-anchored TFPI.
These cells express a lot of TFPI and I would expect to see more
TFPIbeta in the + PIPLC samples than in the - PIPLC samples.
2) The antibodies are diluted 1:500 (ab9881) and 1:1000 (ab66544)
giving a concentration of 1ug/ml in the primary antibody solution.
3) Images are attached in a ppt file. The ab9881 antibody was used
first, then the blot was stripped and ab66544 was used.
4) Yes, there is also a weaker band at 45-46kDa with the AB66544
antibody. I developed the blot after stripping and it was blank so
at least all the secondary antibody was removed indicating the
bands are not leftovers from the ab9881 run.

Read More
Answer

Thank you for your reply and the additional information.

I have a couple more questions as well as a few suggestions. I hope you don't mind.

1) Is the rTFPI that you were showing on the left hand side glycosylated or not?
2) Both antibodies show a strong band and lots of weaker bands, whereas ab66544 is cleaner than ab9881.
3) According to your description of using PI-PLC, it seems that only ab66544 gives you the expected decrease in signal.

Other ways to check if the correct bands are detected is (1) to de-glycosylate the sample and repeat the WB. The MW of the band should shift to 35 kDa for alpha and 29 kDa for beta isoform.

(2) Further using a positive control (e.g. HeLa lysate (ab29545) [https://www.abcam.com/HeLa-Human-epithelial-carcinoma-cell-line-Whole-Cell-Lysate-ab29545.html] or Jurkat cell lysate (ab7899)[https://www.abcam.com/Jurkat-Human-T-cell-lymphoblast-like-cell-line-Whole-Cell-Lysate-ab7899.html]) could be of help to compare your results with the expected results for the antibodies.

(3) Using less primary and maybe also less secondary antibody could help to reduce the background bands, but would not solve the issue of differing band sizes.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Question

I have a question regarding your TFPI
antibodies that I have been using. When I use your C-terminal specific
antibody, AB9881 which should only detect TFPIalpha, I get strongest
bands
at around 45-46kDa. However, when I use the N-terminal specific
antibody AB66544, which should detect both isoforms of TFPI, I get strongest
bands at
about 52kDa (on the same membrane). Literature states that both
TFPIalpha and TFPIbeta migrates at the same molecular weight due to glycosylation
of the proteins, and we have previously seen this in other cells. Weaker
bands at 52/46kDa are seen with the antibodies as well. Do you have any
explanation for this? Which is the correct band?
Also, do you know where the ab48648 anti-TFPI antibody targets TFPI?
Does it detect only TFPIalpha or both isoforms of TFPI?

Read More
Answer

Thank you for contacting us.

Antibody ab9881, detects only the alpha isoform.
Antibody ab66544, would detect both isoforms if they are present
For antibody ab48648, we do not know where the epitope is, thus we cannot predict if either one or both isoforms can be detected. I can check with the lab where they saw a band in WB, though.

Unglycosylated TFPI has a MW of35 kDa for the alpha isoform, and29 kDafor the beta isoform.
The glycoslyation would shift the MW to a higher kDa value.
Glycosylation patterncan also vary based on tissue / species / treatment used.

I'd like to ask you a few more questions to understand your experiment better:
1) What samples are you using?
2) What antibody dilutions are you using?
3) Could you please an image of the WB with both antibodies?
4) For ab66544, do you also see bands at 45-46 kDa?

Assuming that your samples contain both isoforms, then the only explanation for the results you see is that the glycosylation of isoform beta is heavier than for alpha isoform.
If only one isoform is present then this seems to be isoform alpha. For ab66544, we also saw the band at 52 kDa and your results would match our findings.

I'll check with the lab regarding ab48648.

I look forward to hear back from you and assist you further.

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